Fluorescent In-Situ Hybridization of Bladder Microbiome

Five micron paraffin-embedded bladder sections were de-paraffinised in xylene/ethanol. Sections were incubated in lysozyme solution (10 mg/ml, Sigma, France) for 15 minutes at 37°C and exposed to 100 μl of universal bacterial 16 S fluorescent rRNA probe (Eub338, GCTGCCTCCCGTAGGAGT-Cy5’, Eurofins, France) at a concentration of 5 ng/μl, in hybridisation buffer (20 mM Tris-HCl, pH7.4, 0.9 M NaCl, 0.01% SDS) at 46°C for 3 hours [57 (link)]. Sections were then incubated in a 48°C prewarmed saline-sodium citrate wash buffer (30 mM sodium citrate, 300 mM sodium chloride, pH7.4, Invitrogen, France) for 20 minutes. To stain polysaccharide-rich content, sections were counterstained with FITC-conjugated wheat germ agglutinin (Invitrogen, France) at a 1:1000 dilution in PBS buffer for 30 min. Slides were mounted with Fluoroshield containing DAPI. Images were acquired using a confocal laser scanning microscope (Zeiss LSM 710) and final images were processed and edited with the ImageJ software.